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1.
Cell Commun Signal ; 22(1): 235, 2024 Apr 20.
Article in English | MEDLINE | ID: mdl-38643161

ABSTRACT

BACKGROUND: Antral follicles consist of an oocyte cumulus complex surrounding by somatic cells, including mural granulosa cells as the inner layer and theca cells as the outsider layer. The communications between oocytes and granulosa cells have been extensively explored in in vitro studies, however, the role of oocyte-derived factor GDF9 on in vivo antral follicle development remains elusive due to lack of an appropriate animal model. Clinically, the phenotype of GDF9 variants needs to be determined. METHODS: Whole-exome sequencing (WES) was performed on two unrelated infertile women characterized by an early rise of estradiol level and defect in follicle enlargement. Besides, WES data on 1,039 women undergoing ART treatment were collected. A Gdf9Q308X/S415T mouse model was generated based on the variant found in one of the patients. RESULTS: Two probands with bi-allelic GDF9 variants (GDF9His209GlnfsTer6/S428T, GDF9Q321X/S428T) and eight GDF9S428T heterozygotes with normal ovarian response were identified. In vitro experiments confirmed that these variants caused reduction of GDF9 secretion, and/or alleviation in BMP15 binding. Gdf9Q308X/S415T mouse model was constructed, which recapitulated the phenotypes in probands with abnormal estrogen secretion and defected follicle enlargement. Further experiments in mouse model showed an earlier expression of STAR in small antral follicles and decreased proliferative capacity in large antral follicles. In addition, RNA sequencing of granulosa cells revealed the transcriptomic profiles related to defective follicle enlargement in the Gdf9Q308X/S415T group. One of the downregulated genes, P4HA2 (a collagen related gene), was found to be stimulated by GDF9 protein, which partly explained the phenotype of defective follicle enlargement. CONCLUSIONS: GDF9 bi-allelic variants contributed to the defect in antral follicle development. Oocyte itself participated in the regulation of follicle development through GDF9 paracrine effect, highlighting the essential role of oocyte-derived factors on ovarian response.


Subject(s)
Infertility, Female , Mice , Animals , Female , Humans , Infertility, Female/metabolism , Ovarian Follicle/metabolism , Oocytes/chemistry , Oocytes/metabolism , Granulosa Cells/metabolism , Estrogens/metabolism , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/analysis , Growth Differentiation Factor 9/metabolism
2.
J Clin Endocrinol Metab ; 107(8): e3374-e3383, 2022 07 14.
Article in English | MEDLINE | ID: mdl-35511085

ABSTRACT

CONTEXT: The oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) play essential roles in follicle development and oocyte maturation, and aberrant regulation might contribute to the pathogenesis of polycystic ovary syndrome. OBJECTIVE: Are there measurable differences in concentrations of GDF9, BMP15, and the GDF9/BMP15 heterodimer in small antral follicle fluids from women with and without polycystic ovaries (PCO)? DESIGN AND SETTING: Follicle fluids (n = 356) were collected from 4- to 11-mm follicles in unstimulated ovaries of 87 women undergoing ovarian tissue cryopreservation for fertility preservation. PATIENTS: Twenty-seven women with PCO were identified and 60 women without PCO-like characteristics (non-PCO women) were matched according to age and follicle size. MAIN OUTCOME MEASURES: Intrafollicular concentrations of GDF9, BMP15, GDF9/BMP15 heterodimer, anti-Mullerian hormone (AMH), inhibin-A and -B, total inhibin, activin-B and -AB, and follistatin were measured using enzyme-linked immunosorbent assays. RESULTS: The detectability of GDF9, BMP15, and the GDF9/BMP15 heterodimer were 100%, 94.4%, and 91.5%, respectively, and concentrations were significantly negatively correlated with increasing follicle size (P < 0.0001). GDF9 was significantly higher in women with PCO (PCO: 4230 ±â€…189 pg/mL [mean ±â€…SEM], n = 188; non-PCO: 3498 ±â€…199 pg/mL, n = 168; P < 0.03), whereas BMP15 was lower in women with PCO (PCO: 431 ±â€…40 pg/mL, n = 125; non-PCO: 573 ±â€…55 pg/mL, n = 109; P = 0.10), leading to a significantly higher GDF9:BMP15 ratio in women with PCO (P < 0.01). Significant positive associations between BMP15 and AMH, activins, and inhibins in non-PCO women switched to negative associations in women with PCO. CONCLUSIONS: Intrafollicular concentrations of GDF9 and BMP15 varied inversely in women with PCO reflecting an aberrant endocrine environment. An increased GDF9:BMP15 ratio may be a new biomarker for PCO.


Subject(s)
Bone Morphogenetic Protein 15 , Follicular Fluid , Growth Differentiation Factor 9 , Oocytes , Polycystic Ovary Syndrome , Anti-Mullerian Hormone/analysis , Anti-Mullerian Hormone/metabolism , Biomarkers/analysis , Biomarkers/metabolism , Bone Morphogenetic Protein 15/analysis , Bone Morphogenetic Protein 15/metabolism , Female , Follicular Fluid/chemistry , Growth Differentiation Factor 9/analysis , Growth Differentiation Factor 9/metabolism , Humans , Inhibins/metabolism , Oocytes/metabolism , Polycystic Ovary Syndrome/diagnosis , Polycystic Ovary Syndrome/metabolism , Transforming Growth Factor beta/metabolism
3.
J Assist Reprod Genet ; 39(1): 117-125, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34845575

ABSTRACT

PURPOSE: We aimed to evaluate the link between the GDF9 concentration in day 3 human embryo culture medium and embryo quality and viability. METHODS: Two independent, prospective, observational studies were conducted. In study 1, a total of 280 embryos from 70 patients who obtained at least 4 embryos with 6-10 blastomeres (2 transferable and 2 non-transferable embryos) at day 3 were enrolled. In study 2, a total of 119 embryos from 61 patients (29 fully implanted and 32 non-implanted patients) were enrolled. The corresponding GDF9 concentrations in spent culture medium of embryos were quantified by ELISA assay. The expression pattern of GDF9 in human embryos was investigated using Q-PCR and immunofluorescence. RESULTS: GDF9 mRNA and protein were detected from human oocytes to eight-cell embryos and displayed a slow decreasing trend. In study 1, GDF9 concentration in culture medium is lower for transferable embryos compared with non-transferable embryos (331 pg/mL (quartiles: 442, 664 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001), and increased commensurate with the diminution of the embryo quality (P < 0.001). In study 2, significantly lower GDF9 concentration was detected for implanted embryos than non-implanted embryos (331 pg/mL (quartiles: 156, 665 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001). The same trend was found between the embryos that led to live birth and those that failed. CONCLUSION: The GDF9 concentration in culture medium is linked to embryo quality and viability, and exhibited the potential to be a non-invasive biomarker for embryo selection.


Subject(s)
Culture Media/metabolism , Embryonic Development/physiology , Growth Differentiation Factor 9/analysis , Adult , China , Culture Media/pharmacology , Embryo Culture Techniques/methods , Embryo Culture Techniques/statistics & numerical data , Growth Differentiation Factor 9/metabolism , Humans , Prospective Studies
4.
Biol Reprod ; 99(3): 527-535, 2018 09 01.
Article in English | MEDLINE | ID: mdl-29590310

ABSTRACT

Proper development and maturation of oocytes requires interaction with granulosa cells. Previous reports have indicated that mammalian oocytes connect with cumulus cells through gap junctions at the tip of transzonal projections that extend from the cells. Although the gap junctions between oocytes and transzonal projections provide a pathway through which small molecules (<1 kDa) can travel, it is unclear how molecules >1 kDa are transported between the oocytes and cumulus cells. In this study, we presented new connections between oocytes and granulosa cells. The green fluorescein protein Aequorea coerulescens green fluorescein protein (AcGFP1) localizing in oocyte cell membrane, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and dextran conjugates (10,000 MW) injected into the oocytes, which were unable to pass through gap junctions, were diffused from the oocytes into the surrounding granulosa cells through these connections. These connect an oocyte to the surrounding cumulus and granulosa cells by fusing with the cell membranes and forming a large complex during follicle development. Furthermore, we show two characteristics of these connections during follicle development-the localization of growth and differentiation factor-9 within the connections and the dynamics of the connections at ovulation. This article presents for the first time that mammalian oocytes directly connect to granulosa cells by fusing with the cell membrane, similar to that in Drosophila.


Subject(s)
Cell Membrane/physiology , Granulosa Cells/ultrastructure , Membrane Fusion/physiology , Oocytes/ultrastructure , Ovarian Follicle/growth & development , Animals , Cell Membrane/chemistry , Female , Fluorescent Antibody Technique , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/metabolism , Growth Differentiation Factor 9/analysis , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Microscopy, Immunoelectron , Oocytes/growth & development , Ovulation , Tissue Culture Techniques
5.
Reprod Sci ; 22(1): 68-74, 2015 Jan.
Article in English | MEDLINE | ID: mdl-24784715

ABSTRACT

We performed an immunohistochemical (IHC) study to determine the follicular expression of growth differentiation factor 9 (GDF-9), anti-Müllerian hormone (AMH), Kit Ligand (KL), and c-Kit in squirrel monkey ovary. Ovarian tissue fragments from 4 squirrel monkeys were collected by laparotomy and processed for classical histology and IHC. Additionally, follicle development was assessed by Ki67 immunostaining to evaluate proliferative status of granulosa cells. A total of 4025 follicles were examined (1475 for classical histology and 2550 for immunohistochemistry). More than 80% of the evaluated follicles were morphologically normal. The GDF-9 protein was detectable in oocyte cytoplasm from primordial (100%), primary (99.1%), and secondary (100%) follicles. The AMH was not expressed in primordial follicles but just in few primary follicles (13.8%). On the other hand, it was highly expressed in granulosa cells from secondary follicles (67.9%). c-Kit, KL receptor, was found in the oolemma of primordial (100%), primary (100%), and secondary (100%) follicles. The KL expression was observed in oocytes and granulosa cells from primordial (94.9%), primary (91.6%) and secondary follicles (100%). Ki67 immunostaining was observed in granulosa cells from primary (5.7%) and secondary (54.8%) follicles but not in primordial follicles. In conclusion, we described the localization of GDF-9, KL, c-Kit, and Ki67 proteins and confirmed the presence of AMH protein in preantral follicles from squirrel monkey. Our results offer contribution for understanding of folliculogenesis in neotropical nonhuman primates. Moreover, these markers can be used to assess follicular viability and functionality after cryopreservation, transplantation, or in vitro culture of ovarian tissue.


Subject(s)
Anti-Mullerian Hormone/analysis , Cell Proliferation , Growth Differentiation Factor 9/analysis , Immunohistochemistry , Ovarian Follicle/chemistry , Proto-Oncogene Proteins c-kit/analysis , Saimiri/physiology , Stem Cell Factor/antagonists & inhibitors , Age Factors , Animals , Female , Ovarian Follicle/cytology , Saimiri/metabolism
6.
Mol Reprod Dev ; 80(11): 895-907, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23877969

ABSTRACT

Exposure of Siberian hamsters to short photoperiod (SD) inhibits ovarian function, including folliculogenesis, whereas function is restored with their transfer to long photoperiods (LD). To investigate the mechanism of photo-stimulated recrudescence, we assessed key folliculogenic factors-anti-Müllerian hormone (AMH), inhibin-α, growth differentiation factor-9 (GDF9), and bone morphogenic protein-15 (BMP15)-across the estrus cycle and in photo-regressed and recrudescing ovaries. Adult hamsters were exposed to either LD or SD for 14 weeks, which respectively represent functional and regressed ovaries. Select regressed hamsters were transferred back to LD for 2 (post-transfer week 2; PTw2) or 8 weeks (PTw8). Ovaries were collected and fixed in formalin for immunohistochemistry or frozen in liquid nitrogen for real-time PCR. AMH, inhibin-α, GDF9, and BMP15 mRNA and protein were detected in all stages of the estrus cycle. Fourteen weeks of SD exposure increased (P < 0.05) ovarian AMH, GDF9, and BMP15, but not inhibin-α mRNA levels as compared to LD. Transfer of regressed hamsters to stimulatory long photoperiod for 8 weeks returned AMH and GDF9 mRNA levels to LD-treated levels, and further increased mRNA levels for inhibin-α and BMP15. Immunostaining for AMH, inhibin-α, GDF9, and BMP15 proteins was most intense in preantral/antral follicles and oocytes. The overall immunostaining extent for AMH and inhibin-α generally mirrored the mRNA data, though no changes were observed for GDF9 or BMP15 immunostaining. Shifts in mRNA and protein levels across photoperiod conditions suggest possible syncretic roles for these folliculogenic factors in photo-stimulated recrudescence via potential regulation of follicle recruitment, preservation, and development.


Subject(s)
Anti-Mullerian Hormone/analysis , Bone Morphogenetic Protein 15/analysis , Growth Differentiation Factor 9/analysis , Inhibins/analysis , Ovary , Photoperiod , RNA, Messenger/analysis , Animals , Anti-Mullerian Hormone/chemistry , Anti-Mullerian Hormone/genetics , Anti-Mullerian Hormone/metabolism , Bone Morphogenetic Protein 15/chemistry , Bone Morphogenetic Protein 15/genetics , Bone Morphogenetic Protein 15/metabolism , Cricetinae , Estradiol/blood , Female , Growth Differentiation Factor 9/chemistry , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Humans , Immunohistochemistry , Inhibins/chemistry , Inhibins/genetics , Inhibins/metabolism , Ovary/chemistry , Ovary/metabolism , Ovary/radiation effects , Phodopus , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recurrence
7.
Anticancer Res ; 32(10): 4375-83, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23060562

ABSTRACT

BACKGROUND: Growth differentiation factor-9 (GDF9), a member of the bone morphogenetic protein (BMP) family and the transforming growth factor (TGF)-beta superfamily, has recently been implicated in the biological control of cancer cell behaviour. It has also been implicated in the development and spread of solid cancer. However, the role of GDF9 in kidney cancer remains to be investigated. In the present study, the expression of GDF9 in normal and malignant human kidney tissues and its molecular and cellular impact on human kidney cancer cells were investigated. MATERIALS AND METHODS: The expression of GDF9 in human kidney tissues and kidney cancer cell lines (UMRC-2 and CAKI-2) was assessed at both the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. GDF9 overexpression was induced by a mammalian GDF9 expression construct. The effect of GDF9 expression on cellular functions was examined in kidney cancer cells overexpressing GDF9 using a variety of in vitro assays. RESULTS: In normal kidney tissues, stronger staining of GDF9 was seen in renal tubular epithelial cells, both in the cytoplasm and in the nucleus. In contrast, the staining of GDF9 was notably weak or absent in cells of tumour tissues. Human kidney cancer cell lines UMRC-2 and CAKI-2 had lost their GDG-9 expression. Overexpression of GDF9 reduced in vitro invasion and cellular growth and migration of kidney cell lines in vitro. Using the electric cell-substrate sensing (ECIS) method, it was further revealed that overexpression of GDF9 in these cells markedly reduced cellular migration and adhesion. CONCLUSION: Human kidney tumours have a reduced or loss of expression of GDF9. In vitro, GDF9 overexpression suppresses the invasiveness, growth and migration of kidney cancer cells. This suggests that GDF9 is a potential tumour suppressor and may have prognostic and therapeutic implications in human kidney cancer.


Subject(s)
Carcinoma, Renal Cell/pathology , Growth Differentiation Factor 9/biosynthesis , Kidney Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Growth Differentiation Factor 9/analysis , Growth Differentiation Factor 9/genetics , Humans , Immunohistochemistry , Neoplasm Invasiveness , Transfection
8.
Reproduction ; 144(5): 557-67, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22967842

ABSTRACT

The aims were to investigate whether oocyte-secreted growth factors from a high (i.e. rat) and low (i.e. sheep) ovulation rate species could stimulate (3)H-thymidine incorporation in granulosa cells (GC) from antral follicles from the same or across species. Denuded oocytes (DO) were co-incubated with GC with or without specific antibodies to growth differentiating factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15). Co-incubations of DO-GC from the same or across species significantly increased thymidine incorporation in GC with increasing numbers of DO. GDF9 immuno-neutralisation reduced thymidine incorporation in rat GC co-incubated with either rat or ovine DO and in ovine GC co-incubated with ovine or rat DO. BMP15 immuno-neutralisation only reduced thymidine incorporation when ovine DO were co-incubated with either ovine or rat GC. Western blotting of oocytes co-incubated with GC identified GDF9 and BMP15 proteins for sheep and GDF9 protein for rats in oocyte lysates and incubation media. With respect to rat BMP15, a promature protein was identified in the oocyte lysate but not in media. Expression levels of GDF9 relative to BMP15 mRNA in DO co-incubated with GC were highly correlated (R (2)=0.99) within both species. However, the expression ratios were markedly different for the rat and sheep (4.3 vs 1.0 respectively). We conclude that during follicular development, rat oocytes secrete little, if any, BMP15 and that GDF9 without BMP15 can stimulate proliferation of rat and ovine GC. In contrast, ovine oocytes secrete both BMP15 and GDF9, and both were found to stimulate proliferation in ovine and rat GC.


Subject(s)
Bone Morphogenetic Protein 15/physiology , Granulosa Cells/physiology , Growth Differentiation Factor 9/physiology , Oocytes/physiology , Animals , Antibodies, Monoclonal/pharmacology , Bone Morphogenetic Protein 15/analysis , Bone Morphogenetic Protein 15/genetics , Cell Proliferation , Female , Gene Expression , Growth Differentiation Factor 9/analysis , Growth Differentiation Factor 9/genetics , Oocytes/metabolism , Ovulation , RNA, Messenger/analysis , Rats , Sheep , Species Specificity , Thymidine/metabolism , Tritium
9.
J Dairy Sci ; 95(4): 1885-93, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22459835

ABSTRACT

Connection between mastitis and fertility is multifaceted; therefore, several aspects need more elucidation. In particular, the aim was to investigate if naturally occurring chronic mastitis has an effect on ovarian function. At the time of slaughter, a milk sample and both ovaries were collected from 68 cows. The presence and intensity of chronic mastitis was diagnosed by the combined evaluation of bacteriological examination and somatic cell count of the milk of each individual quarter according to the measures of the National Mastitis Council. Animals were divided into 4 groups characterized by a low (n=15), mild (n=14), intense (n=19), or severe (n=16) degree of infection. A count of visible follicles on each ovary was followed by a quantitative analysis of microscopic traits on a selected group of animals (n=16). The latter included the classification and count of the entire preantral follicle population, and the morphometric analysis of the vascular bed extension and connective stroma in the cortical region. Finally, the expression of growth and differentiation factor-9 (GDF-9) was studied. The number of follicles with diameters ranging from 1 to 3 mm and 4 to 7 mm was not affected by the degree of infection. A significant effect of the degree of udder infection was observed on the number of follicles with a diameter larger than 8 mm. Furthermore, the intensity of mastitis had no effect on the number of primordial and primary follicles, but severely affected cows showed a lower number of secondary follicles (0.5±0.1 vs. 0.2±0.03). Quantitative analysis demonstrated a decrease in the density of blood vessels (6.30±1.08 vs. 4.68±0.28) expressed as ratio of vascular bed/total area) and a higher incidence of fibrous stroma (1.60±0.99 vs. 6.04±3.08 expressed as ratio of connective tissue/total area) in the cortical area of the most affected animals. Finally, the level of GDF-9 protein within the oocytes of different follicle size was lower in the animals with the severe form of chronic mastitis (1.34±0.05 vs. 0.78±0.21 expressed as arbitrary units). In conclusion, decreased fertility of cows with chronic mastitis takes place through an effect on the ovary altering the dynamics of folliculogenesis. Within the ovary, this implies a reduction of the vascular bed and an increase in the fibrotic tissue together with a direct effect on oocyte-specific factors as GDF-9, all of which are essential regulatory elements of folliculogenesis.


Subject(s)
Growth Differentiation Factor 9/analysis , Mastitis, Bovine/physiopathology , Ovarian Follicle/physiopathology , Animals , Cattle , Cattle Diseases/etiology , Chronic Disease , Dairying , Female , Infertility, Female/etiology , Infertility, Female/veterinary , Mastitis, Bovine/complications , Mastitis, Bovine/pathology , Oocytes/chemistry , Ovarian Follicle/pathology
10.
Fertil Steril ; 95(7): 2274-8, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21496799

ABSTRACT

OBJECTIVE: To evaluate the association between follicular fluid levels of propeptide and mature forms of growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 with subsequent oocyte and embryo quality. DESIGN: Prospective clinical study. SETTING: University hospital. PATIENT(S): Eighty-one infertile patients who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). INTERVENTION(S): The expression levels of the propeptide and mature forms of follicular fluid GDF9 and BMP15 were determined by western blot analysis. The levels of follicular fluid hormones (FSH, E2, and P) were measured with automated chemiluminescent enzyme immunoassays. MAIN OUTCOME MEASURE(S): The relationships between the levels of GDF9 and BMP15, hormones, oocyte maturation, and embryo quality. RESULT(S): Mature GDF9 levels were significantly correlated with the nuclear maturation of oocytes. The mean mature GDF9 level was 4.87±0.60 in the high-embryo-quality group and 1.45±0.81 in the low-embryo-quality group. There were no statistically significant differences in embryo quality among the patients regarding propeptide GDF9 and BMP15 expression status. There was a negative correlation between follicular fluid levels of P and the mature form of GDF9. CONCLUSION(S): Higher mature GDF9 levels in the follicular fluid were significantly correlated with oocyte nuclear maturation and embryo quality.


Subject(s)
Blastomeres/physiology , Bone Morphogenetic Protein 15/analysis , Fertilization in Vitro , Follicular Fluid/chemistry , Growth Differentiation Factor 9/analysis , Oocyte Retrieval , Oocytes/physiology , Adult , Blotting, Western , Chi-Square Distribution , Cleavage Stage, Ovum , Embryo Culture Techniques , Estradiol/analysis , Female , Follicle Stimulating Hormone, Human/analysis , Hospitals, University , Humans , Immunoenzyme Techniques , Linear Models , Progesterone/analysis , Prospective Studies , Sperm Injections, Intracytoplasmic , Treatment Outcome , Turkey
11.
Reproduction ; 136(2): 195-203, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18469040

ABSTRACT

Growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and anti-Müllerian hormone (AMH) play an important role in the primary to secondary follicle transition and follicle activation in vivo. In organ culture of neonatal mouse ovaries, it was observed that significantly fewer primary follicles develop to the secondary stage. The objectives of this study were: (1) to compare ovarian follicular populations between organ-cultured neonatal mouse ovaries and freshly isolated age-matched control ovaries; (2) to quantify RNA levels of Gdf9, Bmp15, and Amh in cultured primary follicles; and (3) to immunolocalize GDF9 and AMH in cultured ovaries. Ovaries from 3-day-old (PND 3) mice were cultured for 7 or 10 days in the absence or presence of FSH. Follicular populations were counted in freshly isolated 13-day-old (PND 13) ovaries and organ-cultured ovaries. Transcripts were quantified in isolated primary follicles using real-time RT-PCR, and protein expressions were localized using immunohistochemistry. The number of secondary follicles in organ-cultured ovaries was significantly lower than in vivo controls. Gdf9 and Bmp15 mRNA expression levels were similar as in controls. Amh mRNA levels were significantly (P<0.05) lower after day 10 of culture in the absence of FSH. GDF9 and AMH proteins were respectively detected in the oocytes and the granulosa cells (GC) beginning at the primary and primordial stages onward. GDF9 and BMP15 production in cultured primary follicles are not different from in vivo controls; hence abnormal early follicular growth was not related to a deficient transcription of these factors.


Subject(s)
Anti-Mullerian Hormone/analysis , Bone Morphogenetic Protein 15/analysis , Growth Differentiation Factor 9/analysis , Ovarian Follicle/chemistry , Animals , Female , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Tissue Culture Techniques
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